The goal of this research proposal is to develop an understanding of the molecular mechanisms by which steroid hormones regulate gene expression during development and differentiation. We will pursue this goal by: (1) observing and characterizing the primary events which occur during the interaction of steroid hormone-receptor complexes with the nucleus and (2) reconstituting the transcription and regulation of steroid-responsive genes in a cell-free system. We will focus our attention on the chick transferrin gene as a model for hormonal regulation of gene expression. The transferrin gene is induced by both estrogen and progesterone in chick oviduct and the entire response can be reproduced under well-defined tissue culture conditions. Preliminary experiments suggest that protein intermediates, protein modifications (acetylation and phosphorylation), and polypeptide hormones (insulin) play an important role in the steroid response, and we plan to explore these observations at the molecular level. In addition, the transferrin gene is active in the liver where it is modulated by iron deficiency as well as hormones; this gene provides an excellent opportunity to study the mechanisms which program a specific gene during differentiation. The reconstitution of an active cell-free transcription system in which transferrin gene regulation can be studied directly will be approached by using recombinant DNA containing the genomic transferrin gene, purified RNA polymerase II, and cytoplasmic or nuclear protein fractions. The long-range goal is to identify and purify the regulatory components which are involved in the hormone-mediated response and to study the interaction of these molecules with the gene.